To enhance site-specific intracellular delivery against folate receptor, heparin-PEG-folate (H-PEG-F) containing succinylated-heparin conjugated with folate via PEG 1000/3000 spacers has been prepared. Due to covalent strategy, H-PEG-F displays amphiphilic property, which is capable of entrapping a hydrophobic agent, like taxol, to form heparin-PEG-folate-taxol nanoparticles (H-PEG-F-T NPs) in aqueous solution. Hydrophobic agents can be entrapped within the core, while the H-PEG-F conjugates can stabilize the nanoparticles with exposing folate moieties on the surface. The structure of carrier and naoparticles has been characterized by(1)H NMR, and the content of folate and taxol has been quantitatively analyzed by UV method. The morphology and size of H-PEG-F-T NPs have been measured by field emission scanning electron microscopy (FESEM) and dynamic lighting scatter (DLS). All the NPs are in spherical shape and the sizes are less than 200 nm. The sizes of the NPs increases with increasing PEG segment length. By employing the flow cytomery method, the extent of cellular uptake has been comparatively evaluated under various conditions. The results of cellular uptake demonstrate that the cellular uptake of the carrier and the NPs is exceedingly higher for KB-3-1 cells (folate receptor overexpressing cell line) than for A549 cells (folate receptor deficiency cell line); H-PEG-F-T NPs show far greater extent of cellular uptake than that of H-PEG-F conjugates against A549 cells; when the content of folate is fixed at the same value, the extent of cellular uptake for the carrier and NPs ascends with the increase of PEG chain length against KB-3-1 cells. It suggests folate-receptor-mediated endocytosis and formation of nanoparticle and spacer length are considered to coaffect the cellular uptake efficiency of H-PEG-F-T NPs and H-PEG-F conjugates. Flow cytometry analysis depicts that KB-3-1 cells treated with H-PEG-F-T are arrested in the G(2)/M phase of the cell cycle, which states the similar inhibition mechanism as taxol. The strategy based on the formation of H-PEG-F-T NPs could be potentially applied for cancer cell targeted delivery of various therapeutic agents.